Recombinant Viral-Based Malaria Vaccines

ABSTRACT

Described are vaccines against malarial infections, which are based on recombinant viral vectors, such as alpha viruses, adenoviruses, or vaccinia viruses. The recombinant viral-based vaccines can be used to immunize against different  Plasmodium  infections, such as infections by  P. falciparum  or  P. yoelii . Codon-optimized circumsporozoite genes are disclosed. Replication-defective adenoviruses may be used, derived from serotypes that encounter low titers of neutralizing antibodies. Also described is the use of different adenoviral serotypes that are administered to elicit a strong immune response, either in single vaccination set-ups or in prime-boost set-ups in which compositions based on different serotypes can be applied.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 12/928,044, filed Dec. 1, 2010, which is a continuation of U.S.Ser. No. 11/975,396, filed Oct. 18, 2007, now U.S. Pat. No. 7,867,764,issued Jan. 11, 2011, which is a divisional of U.S. Ser. No. 11/607,366,filed Dec. 1, 2006, now U.S. Pat. No. 7,387,894, issued Jun. 17, 2008,which is a divisional of U.S. patent application Ser. No. 11/143,986,filed Jun. 2, 2005, now U.S. Pat. No. 7,300,657, issued Nov. 27, 2007,which is a continuation of PCT International Patent Application No.PCT/EP2003/051019, filed on Dec. 16, 2003, designating the United Statesof America, and published, in English, as PCT International PublicationNo. WO 2004/055187 A 1 on Jul. 1, 2004, which, in turn, claims priorityunder Article 8 of the PCT to PCT International Patent Application No.PCT/EP03/50222, filed on Jun. 12, 2003, which, in turn, claims priorityto European Patent Application Serial No. 02102781.8 filed Dec. 17,2002, the disclosures of each of which are hereby incorporated herein intheir entirety by this reference.

TECHNICAL FIELD

The disclosure generally relates to the field of medicine andbiotechnology. More particularly, the disclosure relates to the use of arecombinantly produced viral vector as a carrier of an antigenicdeterminant selected from a group of malaria pathogens for thedevelopment of a vaccine against malaria infections.

BACKGROUND

Malaria currently represents one of the most prevalent infections intropical and subtropical areas throughout the world. Per year, malariainfections lead to severe illnesses in hundreds of million individualsworldwide, while it kills 1 to 3 million people, primarily in developingand emerging countries every year. The widespread occurrence andelevated incidence of malaria are a consequence of the increasingnumbers of drug-resistant parasites and insecticide-resistant parasitevectors. Other factors include environmental and climatic changes, civildisturbances, and increased mobility of populations.

Malaria is caused by the mosquito-borne hematoprotozoan parasitesbelonging to the genus Plasmodium. Four species of Plasmodium protozoa(P. falciparum, P. vivax, P. ovale and P. malariae) are responsible forthe disease in man; many others cause disease in animals, such as P.yoelii and P. berghei in mice. P. falciparum accounts for the majorityof infections and is the most lethal type (“tropical malaria”). Malariaparasites have a life cycle consisting of several stages. Each stage isable to induce specific immune responses directed against thecorresponding occurring stage-specific antigens.

Malaria parasites are transmitted to man by several species of femaleAnopheles mosquitoes. Infected mosquitoes inject the “sporozoite” formof the malaria parasite into the mammalian bloodstream. Sporozoitesremain for a few minutes in the circulation before invading hepatocytes.At this stage, the parasite is located in the extra-cellular environmentand is exposed to antibody attack, mainly directed to the“circumsporozoite” (CS) protein, a major component of the sporozoitesurface. Once in the liver, the parasites replicate and develop intoso-called “schizonts.” These schizonts occur in a ratio of up to 20,000per infected cell. During this intra-cellular stage of the parasite,main players of the host immune response are T-lymphocytes, especiallyCD8+ T-lymphocytes (Romero et al. 1998). After about one week of liverinfection, thousands of so-called “merozoites” are released into thebloodstream and enter red blood cells, becoming targets ofantibody-mediated immune response and T-cell secreted cytokines. Afterinvading erythrocytes, the merozoites undergo several stages ofreplication and transform into so-called “trophozoites” and intoschizonts and merozoites, which can infect new red blood cells. Thisstage is associated with overt clinical disease. A limited amount oftrophozoites may evolve into “gametocytes,” which is the parasite'ssexual stage. When susceptible mosquitoes ingest erythrocytes,gametocytes are released from the erythrocytes, resulting in severalmale gametocytes and one female gametocyte. The fertilization of thesegametes leads to zygote formation and subsequent transformation intoookinetes, then into oocysts, and finally into salivary glandsporozoites.

Targeting antibodies against gametocyte stage-specific surface antigenscan block this cycle within the mosquito mid gut. Such antibodies willnot protect the mammalian host but will reduce malaria transmission bydecreasing the number of infected mosquitoes and their parasite load.

Current approaches to malaria vaccine development can be classifiedaccording to the different stages in which the parasite can exist, asdescribed above. Three types of possible vaccines can be distinguished:

-   -   Pre-erythrocytic vaccines, which are directed against        sporozoites and/or schizont-infected cells. These types of        vaccines are primarily CS-based, and should ideally confer        sterile immunity, mediated by humoral and cellular immune        response, preventing malaria infection.    -   Asexual blood-stage vaccines, which are designed to minimize        clinical severity. These vaccines should reduce morbidity and        mortality and are meant to prevent the parasite from entering        and/or developing in the erythrocytes.    -   Transmission-blocking vaccines, which are designed to hamper the        parasite development in the mosquito host. This type of vaccine        should favor the reduction of population-wide malaria infection        rates.

Next to these vaccines, the feasibility of developing malaria vaccinesthat target multiple stages of the parasite life cycle is being pursuedin so-called multi-component and/or multi-stage vaccines. Currently, nocommercially available vaccine against malaria is available, althoughthe development of vaccines, against malaria has already been initiatedmore than 30 years ago: immunization of rodents, non-human primates andhumans with radiation-attenuated sporozoites conferred protectionagainst a subsequent challenge with sporozoites (Nussenzweig et al.1967; Clyde et al. 1973). However, the lack of a feasible large-scaleculture system for the production of sporozoites prevents the widespreadapplication of such vaccines.

To date, the most promising vaccine candidates tested in humans havebeen based on a small number of sporozoite surface antigens. The CSprotein is the only P. falciparum antigen demonstrated to consistentlyprevent malaria when used as the basis of active immunization in humansagainst mosquito-borne infection, albeit it at levels that is ofteninsufficient. Theoretical analysis has indicated that the vaccinecoverage, as well as the vaccine efficiency, should be above 85% or,otherwise, mutants that are more virulent may escape (Gandon et al.2001).

One way of inducing an immune response in a mammal is by administeringan infectious carrier that harbors the antigenic determinant in itsgenome. One such carrier is a recombinant adenovirus, which has beenreplication-defective by removal of regions within the genome that arenormally essential for replication, such as the E1 region. Examples ofrecombinant adenoviruses that comprise genes encoding antigens are knownin the art (PCT International Patent Publication WO 96/39178), forinstance, HIV-derived antigenic components have been demonstrated toyield an immune response if delivered by recombinant adenoviruses (WO01/02607 and WO 02/22080). Also for malaria, recombinantadenovirus-based vaccines have been developed. These vectors express theentire CS protein of P. yoelii, which is a mouse-specific parasite, andthese vectors have been shown to be capable of inducing sterile immunityin mice in response to a single immunizing dose (Bruña-Romero et al.2001a). Furthermore, a similar vaccine vector using CS from P. bergheiwas recently shown to elicit long-lasting protection when used in aprime-boost regimen, in combination with a recombinant vaccinia virus(Gilbert et al. 2002) in mice. It has been demonstrated that CD8+T-cells primarily mediate the adenovirus-induced protection. It isunlikely the P. yoelii- and P. berghei-based adenoviral vectors wouldwork well in humans, since the most dramatic malaria-related illnessesin humans are not caused by these two parasites. Moreover, it ispreferred to have a vaccine which is potent enough to generatelong-lasting protection after one round of vaccination, instead ofmultiple vaccination rounds using either naked DNA injections and/orvaccinia-based vaccines as boosting or priming agents.

Despite all efforts to generate a vaccine that induces an immuneresponse against a malaria antigenic determinant and protects fromillnesses caused by the malaria parasite, many vaccines do not fulfillall requirements as described above. Whereas some vaccines fail to givea protective efficiency of over 85% in vaccinated individuals, othersperform poorly in areas, such as, production or delivery to the correctcells of the host immune system.

BRIEF SUMMARY OF THE INVENTION

Described are different kinds of replication-defective recombinant viralvectors comprising a heterologous nucleic acid encoding an antigenicdeterminant of several Plasmodium protozoa. The viral vectors maycomprise nucleic acids encoding the circumsporozoite (CS) protein of P.falciparum and P. yoelii. The viral vector may be an adenovirus, e.g.,based on a serotype that is efficient in delivering the gene ofinterest, that encounters low numbers of neutralizing antibodies in thehost and that binds to the relevant immune cells in an efficient manner.In certain embodiments, the CS protein is generated such that it willgive rise to a potent immune response in mammals, e.g., humans. In oneaspect, the expression of the protein is elevated due to codonoptimization and thus altering the codon usage such that it fits thehost of interest. The novel CS proteins of the invention are depicted inFIGS. 1A (SEQ ID NO:3 of the incorporated herein SEQUENCE LISTING), 2A(SEQ ID NO:6) and 3A (SEQ ID NO:9), while the codon-optimized genesencoding the proteins are depicted in FIGS. 1B (SEQ ID NO:1), 2B (SEQ IDNO:4) and 3B (SEQ ID NO:7), respectively.

Also described are vaccine compositions comprising areplication-defective recombinant viral vector as described herein and apharmaceutically acceptable carrier, further comprising, e.g., anadjuvant. Furthermore, also described is the use of a vaccinecomposition as disclosed herein in the therapeutic, prophylactic ordiagnostic treatment of malaria.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the newly synthesized clone 02-148 (pCR-script.Pf), whichis based on a range of known Plasmodium falciparum genes and whichencodes the novel circumsporozoite protein (A) (SEQ ID NO:3), plus thecodon-optimized nucleic acid molecule (B) (SEQ ID NO:1). SEQ ID NO:2 isthe translated protein product translated from the Coding Sequence ofSEQ ID NO:1 as generated by PatentIn 3.1. SEQ ID NO:2 is identical toSEQ ID NO:3 in content.

FIG. 2 shows the amino acid molecule (A) (SEQ ID NO:6) and nucleic acidmolecule (B) (SEQ ID NO:4) of synthetic clone named 02-659 (pf-aa-sub),which is the circumsporozoite gene of the P. falciparum strain 3D7,lacking the C-terminal 14 amino acids. SEQ ID NO:5 is the translatedprotein product translated from the Coding Sequence of SEQ ID NO:4 asgenerated by PatentIn 3.1. SEQ ID NO:5 is identical to SEQ ID NO:6 incontent.

FIG. 3 shows the amino acid molecule (A) (SEQ ID NO:9) and nucleic acidmolecule (B) (SEQ ID NO:7) of the codon-optimized circumsporozoite geneof P. yoelii. SEQ ID NO:8 is the translated protein product translatedfrom the Coding Sequence of SEQ ID NO:7 as generated by PatentIn 3.1.SEQ ID NO:8 is identical to SEQ ID NO:9 in content.

FIG. 4 shows the (A) cellular immune response and the (B) humoral immuneresponse in mice upon immunization with Ad5- and Ad35-based vectorsharboring the P. yoelii circumsporozoite gene, administered via tworoutes: intramuscular and subcutaneous in different doses.

FIG. 5 shows the inhibition in mice of a P. yoelii sporozoite challengefollowing immunization with Ad5- and Ad35-based vectors harboring the P.yoelii circumsporozoite gene, administered in different doses, depictedin percentage of inhibition (A), and in the presence ofparasite-specific RNA molecules in the liver (B).

FIG. 6 shows the cellular immune response raised by immunization with anAd5-based vector harboring the full-length P. falciparumcircumsporozoite gene and two deletion mutants, administered indifferent doses.

DETAILED DESCRIPTION OF THE INVENTION

Described is the use of recombinant viruses as carriers of certainspecific antigenic determinants selected from a group of malariaantigens. In various embodiments, provided is a solution to at least apart of the problems outlined above for existing vaccines againstmalaria.

Also described is a replication-defective recombinant viral vectorcomprising a heterologous nucleic acid encoding an antigenic determinantof P. falciparum. In certain embodiments, the viral vector is anadenovirus, an alphavirus or a vaccinia virus. In certain embodiments,the viral vector is an adenovirus, wherein the adenovirus may be derivedfrom a serotype selected from the group consisting of Ad5, Ad11, Ad26,Ad34, Ad35, Ad48, Ad49, and Ad50. In one aspect, thereplication-defective recombinant viral vector as described hereincomprises an antigenic determinant that is the circumsporozoite (CS)protein or an immunogenic part thereof. The heterologous nucleic acidmay be codon optimized for elevated expression in a mammal, e.g., ahuman. Codon optimization is based upon the required amino acid content,the general optimal codon usage in the mammal of interest and a numberof provisions of aspects that should be avoided to ensure properexpression. Such aspects may be splice donor or acceptor sites, stopcodons, Chi-sites, poly(A) stretches, GC- and AT-rich sequences,internal TATA boxes, etc.

In certain embodiments, the disclosure relates to areplication-defective recombinant viral vector as described herein,wherein the adenine plus thymine content in the heterologous nucleicacid, as compared to the cytosine plus guanine content, is less than87%, preferably less than 80%, more preferably less than 59% and mostpreferably equal to approximately 45%. The invention provides, incertain embodiments, a replication-defective recombinant viral vector,wherein the circumsporozoite protein is the circumsporozoite protein asdepicted in FIG. 1A and in another embodiment, a codon-optimizedheterologous nucleic acid as depicted in FIG. 1B. The proteins can be ina purified form, but also expressed in vivo from nucleic acid deliveryvehicles such as the recombinant viral vectors of the invention. In apurified form, such proteins can be applied in other types of vaccines,wherein the protein is, for instance, enclosed in liposomes or othercarriers used in the art. The nucleic acid can be cloned into othervectors than as disclosed herein, but also be applied as naked DNA inother vaccine settings.

In another embodiment, the invention relates to a replication-defectiverecombinant viral vector as described herein, wherein thecircumsporozoite protein, or the immunogenic part thereof, is lacking afunctional GPI anchor sequence.

Apart from the use of new genes and proteins that can be applied for usein humans, also disclosed are novel genes that may be used in humans aswell as in other mammals. Therefore, the disclosure also relates to areplication-defective recombinant viral vector comprising a heterologousnucleic acid encoding the circumsporozoite protein of Plasmodium yoelii,wherein the nucleic acid is codon optimized for elevated expression in amammal.

In certain embodiments, the viral vector is an adenovirus, analphavirus, or a vaccinia virus, and preferably a recombinantadenovirus, which may be selected from the group consisting of Ad5,Ad11, Ad26, Ad34, Ad35, Ad48, Ad49, and Ad50. As in P. falciparum, it isalso for P. yoelii preferred to use a codon-optimized gene for properexpression in the host of interest. Therefore, in certain embodiments,the adenine plus thymine content in the nucleic acid, as compared to thecytosine plus guanine content, is less than 87%, preferably less than80%, more preferably less than 59% and most preferably equal toapproximately 45%.

Also provided is a replication-defective recombinant viral vector asdescribed herein, wherein the circumsporozoite protein is thecircumsporozoite protein as depicted in FIG. 3A, while in anotherembodiment, a replication-defective recombinant viral vector isprovided, wherein the nucleic acid is the nucleic acid depicted in FIG.3B. In one aspect, the circumsporozoite protein, or the immunogenic partthereof, may lack a functional GPI anchor sequence.

Also disclosed is an isolated nucleic acid encoding a circumsporozoiteprotein of Plasmodium falciparum as depicted in FIG. 1B, wherein thenucleic acid is codon optimized, and to an isolated nucleic acidencoding a circumsporozoite protein of P. falciparum strain 3D7, asdepicted in FIG. 2B, wherein the nucleic acid is codon optimized. Suchisolated nucleic acids can be applied in subcloning procedures for thegeneration of other types of viral-based vaccines, apart from the typesas disclosed herein. Furthermore, such isolated nucleic acids can beused for naked DNA vaccines or in cloning procedures to generate vectorsfor in vitro production of the encoded protein, which, in itself, can befurther used for vaccination purposes and the like. The production canbe in all kinds of systems, such as bacteria, yeasts or mammalian cellsknown in the art.

In another embodiment, an isolated nucleic acid encoding acircumsporozoite protein of P. yoelii as depicted in FIG. 3B isprovided, wherein the nucleic acid is codon optimized. Furthermore, avaccine composition comprising a replication-defective recombinant viralvector as described herein and a pharmaceutically acceptable carrier isprovided. Pharmaceutically acceptable carriers are well known in the artand used extensively in a wide range of therapeutic products.Preferably, carriers are applied that work well in vaccines. Morepreferred are vaccines further comprising an adjuvant. Adjuvants areknown in the art to further increase the immune response to an appliedantigenic determinant. The invention also relates to the use of avaccine composition as described herein in the therapeutic, prophylacticor diagnostic treatment of malaria.

Another embodiment relates to a method of treating a mammal for amalaria infection or preventing a malaria infection in a mammal, themethod comprising (in either order or simultaneously) the steps ofadministering a vaccine composition as described herein andadministering a vaccine composition comprising at least one purifiedmalaria-derived protein or peptide. Also described is a method oftreating a mammal for a malaria infection or preventing a malariainfection in a mammal, the method comprising (in either order orsimultaneously) the steps of administering a vaccine compositioncomprising a replication-defective recombinant viral vector comprising amalaria circumsporozoite antigen as described herein and administering avaccine composition comprising a replication-defective recombinant viralvector comprising another antigen, such as LSA-1 or LSA-3 as describedherein.

The advantages of the invention are multi-fold. Next to the knowledgethat recombinant viruses, such as recombinant adenoviruses, can beproduced to very high titers using cells that are considered safe andthat can grow in suspension to very high volumes, using medium that doesnot contain any animal- or human-derived components, the inventioncombines these features with a vector harboring the circumsporozoitegene of P. falciparum. P. falciparum is the parasite that causestropical malaria. Moreover, the gene has been codon optimized to give anexpression level that is suitable for giving a proper immune response inhumans. The invention provides a vaccine against malaria infections,making use of, for instance, adenoviruses that do not encounter hightiters of neutralizing antibodies. Examples of such adenoviruses areserotype 11 and 35 (Ad11 and Ad35, see WO 00/70071 and WO 02/40665).

The nucleic acid content between the malaria-causing pathogen, such asP. falciparum, and the host of interest, such as Homo sapiens, is verydifferent. Now provided is a solution to some of the disadvantages ofvaccines known in the art, such as, expression levels that are too lowto elicit a significant immune response in the host of interest, e.g.,humans.

Recombinant viral vectors have been used in vaccine set-ups. This hasbeen demonstrated for vaccinia-based vaccines and for adenovirus-basedvaccines. Moreover, a platform based on alphaviruses is being developedfor vaccines as well. In certain embodiments, the invention relates tothe use of recombinant adenoviruses that are replication defectivethrough removal of at least part of the E1 region in the adenoviralgenome, because the E1 region is required for replication,transcription, translation and packaging processes of newly madeadenoviruses. E1-deleted vectors are generally produced on cell linesthat complement for the deleted E1 functions. Such cell lines and theuse thereof for the production of recombinant viruses have beendescribed extensively and are well known in the art. Preferably, PER.C6®producer cells, as represented by the cells deposited under ECACC no.96022940 at the European Collection of Animal Cell Cultures (ECACC) atthe Centre for Applied Microbiology and Research (CAMR, UK), are beingused in the cell line to prevent the production of replication-competentadenoviruses (“rca”). In another preferred embodiment, cells are beingapplied that support the growth of recombinant adenoviruses other thanthose derived from adenovirus serotype 5 (“Ad5”). Reference is made topublications WO 97/00326, WO 01/05945, WO 01/07571, WO 00/70071, WO02/40665 and WO 99/55132, for methods and means to obtain rca-freeadenoviral stocks for Ad5, as well as for other adenovirus serotypes.

Adenoviral-based vectors that have been used in the art mainly involvedthe use of Ad5 vectors. However, as has been described (WO 00/03029, WO02/24730, WO 00/70071, WO 02/40665 and in other reports in the art),administration of Ad5 and efficient delivery to the target cells ofinterest responsible for sufficient immunogenic responses, is hamperedby the presence of high titers of neutralizing antibodies circulating inthe bloodstream if a subject previously encountered an Ad5 infection. Ithas been investigated what serotypes are better suited for therapeuticuse and it turned out that a limited number of serotypes encounteredneutralizing antibodies in only a small percentage of individuals in thehuman population. These experiments have been described in WO 00/70071.Therefore, in certain embodiments, the invention relates to the use ofadenovirus serotype 11, 26, 34, 35, 48 and 50 and, more preferably, toAd11 and Ad35, since these serotypes encountered no neutralizingantibodies in the vast majority of tested samples.

Apart from avoiding the presence of neutralizing antibodies directedagainst certain serotypes, it might also be beneficial to target thereplication-deficient recombinant viral vectors to a certain subset ofcells involved in the immune response. Such cells are, for instance,dendritic cells. It was found that certain adenovirus serotypes, such asAd16, Ad35, and Ad50, carry capsid proteins that specifically bind tocertain receptors present on dendritic cells (WO 02/24730). Ad5 is aserotype that is mainly homing to the liver, which may be a disadvantageif sufficient numbers of viral particles should infect cells of theimmune system. It was found that at least in in vitro experiments, someof the serotypes different from Ad5 could infect dendritic cellsmulti-fold better than Ad5, suggesting that also in vivo the delivery tosuch cells is more efficient. It still remains to be seen whether thisin vitro to in vivo translation holds up and if serotypes other than Ad5will give rise to the required protection level. It is also part of theinvention to provide the serotypes of choice, as far as neutralizingantibodies are concerned, with capsid proteins, such as the fiber or apart thereof from a serotype that is able to selectively recognizedendritic cells. It must be noted here that in the published documentsWO 00/03029, WO 02/24730, WO 00/70071, and WO 02/40665, Ad50 wasmistakenly named Ad51. The Ad51 serotype that was referred to in thementioned publications is the same as serotype Ad50 in a publication byDe Jong et al. (1999), wherein it was denoted as a B-group adenovirus.For the sake of clarity, Ad50 as used herein, is the B-group Ad50serotype as mentioned by De Jong et al. (1999).

It is now known that a first administration with a specific adenoviralserotype elicits the production of neutralizing antibodies in the hostagainst that specific vector. Thus, it is desirable to use in asubsequent setting (a follow-up boost or in the administration ofanother, non-related vaccine) a composition based on a differentadenovirus serotype, which is not neutralized by antibodies raised inthe first administration. Therefore, the invention further relates tomethods for vaccinating mammalian individuals in which a priming vaccinecomposition comprises a replication-defective recombinant adenovirus ofa first serotype, while in a boosting vaccine composition, areplication-defective recombinant adenovirus of a second serotype areused. Prime/boost settings have been described in more detail ininternational patent applications PCT/NL02/00671 and PCT/EP03/50748 (notpublished). These applications relate to the use of a recombinantadenovirus vector of a first serotype for the preparation of amedicament for the treatment or prevention of a disease in a human oranimal treated with a recombinant adenovirus vector of a secondserotype, wherein the first serotype is different from the secondserotype, and wherein the first serotype is selected from the groupconsisting of: Ad11, Ad26, Ad34, Ad35, Ad46, and Ad49, and wherein thesecond serotype is preferably adenovirus serotype 5. Thus, it relates tothe use of different adenoviral serotypes that encounter lowpre-existing immunities in subjects that are to be treated. Preferredexamples of such serotypes are the recombinant mentioned, wherein Ad5 isnot excluded for individuals that have never experienced an Ad5infection. The settings described and claimed in the applicationsmentioned above relate to the use of adenoviral vectors carryingtransgenes, such as those from measles, or gag from HIV (for treatmentof humans) or SIV (for treatment and studies in monkeys).

One non-limiting example of a prime-boost set-up towards Malaria is asetting in which, next to different adenovirus serotypes, differentantigenic determinants may also be used. One non-limiting example of anantigen different from CS is the Liver-Specific Antigen 1 (LSA-1, Kurtiset al. 2001). Such set-ups are at least for one reason useful, namely,that the CS antigen is expressed mainly during the blood stage of theparasite, while its expression goes down in the liver stage. For LSA-1,this situation is more or less the opposite; it is expressed to lowlevels during the blood stage, but is highly expressed during the liverstage. Although one could use both antigens in subsequentadministrations, it may also be used at the same time to provideprotection against the parasite at the blood stage as well as at theliver stage. In a further embodiment of the invention, both antigens maybe delivered by one adenovirus serotype (either cloned together in thesame vector or separately in separate vectors of the same serotype). Inanother embodiment, both antigens are delivered by different serotypesthat may be delivered at the same time or separately in time, forinstance, in a prime-boost setting. The vaccines hereof may also be usedin settings in which prime-boosts are being used in combination withnaked DNA or other delivery means, unrelated to thereplication-defective viral vectors hereof, such as purified proteins orpeptides. Examples of such proteins that may be used in prime-boosts(Ad/protein; protein/Ad; protein/Ad/Ad; Ad/protein/Ad; Ad/Ad/protein,etc) are CS, LSA-1, LSA-3, MSP-1, MSP-119, MSP-142 (see below), or thehepatitis B particles containing and CS-derived vaccine compositionknown as RTS,S (see Gordon et al. 1995, U.S. Pat. No. 6,306,625 and WO93/10152).

Although the invention is exemplified herein with the use ofadenoviruses, it is to be understood that the invention is by no meansintended to be limited to adenoviruses but also relates to the use ofother recombinant viruses as delivery vehicles. Examples of viruses thatcan also be used for administering the antigenic determinants of theinvention are poxviruses (vaccinia viruses, such as MVA) andflaviviruses such as alphaviruses. Non-limiting examples of alphavirusesthat may be applied for delivering the immunogenic Plasmodium componentsof the invention are: Ndumu virus, Buggy Creek virus, Highland J. virus,Fort Morgan virus, Babanki virus, Kyzylagach virus, Una virus, Auravirus, Whataroa virus, Bebaru virus, South African Arbovirus No. 86,Mayaro virus, Sagiyama virus, Getah virus, Ross River virus, BarmahForest virus, Chikungunya virus, O'nyong-nyong virus, Western EquineEncephalitis virus (WEE), Middelburg virus, Everglades virus, EasternEncephalitis virus (EEE), Mucambo virus and Pixuna virus. Preferably,when an alphavirus is the virus of choice, Semliki Forest Virus, Sindbisvirus or Venezuelan Equine Encephalitis virus are applied.

A sequence is “derived” as used herein if a nucleic acid can be obtainedthrough direct cloning from wild-type sequences obtained from wild-typeviruses, while they can, for instance, also be obtained through PCR byusing different pieces of DNA as a template. This means also that suchsequences may be in the wild-type form as well as in altered form.Another option for reaching the same result is through combiningsynthetic DNA. It is to be understood that “derived” does notexclusively mean a direct cloning of the wild-type DNA. A person skilledin the art will also be aware of the possibilities of molecular biologyto obtain mutant forms of a certain piece of nucleic acid. The terms“functional part, derivative and/or analogue thereof” are to beunderstood as equivalents of the nucleic acid they are related to. Aperson skilled in the art will appreciate the fact that certaindeletions, swaps, (point) mutations, additions, etc., may still resultin a nucleic acid that has a similar function as the original nucleicacid. It is, therefore, to be understood that such alterations that donot significantly alter the functionality of the nucleic acids arewithin the scope of the invention. If a certain adenoviral vector isderived from a certain adenoviral serotype of choice, it is also to beunderstood that the final product may be obtained through indirect ways,such as direct cloning and synthesizing certain pieces of genomic DNA,using methodology known in the art. Certain deletions, mutations andother alterations of the genomic content that do not alter the specificaspects of the invention are still considered to be part of theinvention. Examples of such alterations are, for instance, deletions inthe viral backbone to enable the cloning of larger pieces ofheterologous nucleic acids. Examples of such mutations are, forinstance, E3 deletions or deletions and/or alterations in the regionscoding for the E2 and/or E4 proteins of adenovirus. Such changes appliedto the adenoviral backbone are known in the art and often applied, sincespace is a limiting factor for adenovirus to be packaged; this is amajor reason to delete certain parts of the adenoviral genome. Otherreasons for altering the E2, E3 and/or E4 regions of the genome may berelated to stability or integrity of the adenoviral vector as, forinstance, described in international patent applications PCT/NL02/00280,PCT/EP03/50125, PCT/NL02/00281, PCT/EP03/50126 (non published). Theseapplications relate, amongst others, to the use of an E4orf6 gene from aserotype from one subgroup in the backbone of an adenovirus from anothersubgroup to ensure compatibility between the E4orf6 activity and theE1B-55K activity during replication and packaging in a packaging cellline. They further relate to the use of a proper functioning pIXpromoter for obtaining higher pIX expression levels and a more stablerecombinant adenoviral vector.

“Replication defective” as used herein means that the viral vectors donot replicate in non-complementing cells. In complementing cells, thefunctions required for replication and, thus, production of the viralvector, are provided by the complementing cell. Thereplication-defective viral vectors of the invention do not harbor allelements enabling replication in a host cell other than a complementingcell.

“Heterologous” as used herein in conjunction with nucleic acids meansthat the nucleic acid is not found in wild-type versions of the viralvectors in which the heterologous nucleic acid is cloned. For instance,in the case of adenoviruses, the heterologous nucleic acid that iscloned in the replication-defective adenoviral vector, is not anadenoviral nucleic acid.

“Antigenic determinant” as used herein means any antigen derived from apathogenic source that elicits an immune response in a host towardswhich the determinant is delivered (administered). Examples of antigenicdeterminants of Plasmodium that can be delivered by using thereplication-defective recombinant viruses of the invention are thecircumsporozoite protein, the SE36 polypeptide, the merezoite surfaceprotein 119 kDa C-terminal polypeptide (MSP-119), MSP-1, MSP-142,Liver-Stage Antigen 1 or 3 (LSA-1 or -3), or a fragment of any of theaforementioned. In a preferred aspect, the invention relates to thecircumsporozoite (CS) protein from P. falciparum.

“Codon-optimized” as used herein means that the nucleic acid content hasbeen altered to reach sufficiently high expression levels of the proteinof interest in a host of interest to which the gene encoding the proteinis delivered. “Sufficiently high expression levels” in this contextmeans that the protein levels should be high enough to elicit an immuneresponse in the host in order to give protection to a malaria-inducingparasite that may enter the treated host before or after treatment. Itis known in the art that some vaccines give an immune response inhumans, through which approximately 60% of the vaccinated individualsare protected against illnesses induced by subsequent challenges withthe pathogen (e.g., sporozoites). Therefore, the expression levels areconsidered to be sufficient if 60% or more of the treated individualsare protected against subsequent infections. It is believed that withthe combinations of adenoviral aspects that can be applied and thechoice of antigen as disclosed herein, such percentages may be reached.Preferably, 85% of the individuals are protected, while it is mostpreferred to have protection to a subsequent challenge in more than 90%of the vaccinated hosts. The nucleic acids disclosed in the inventionare codon optimized for expression in humans. According to Narum et al.(2001), the content of adenine plus thymine (A+T) in DNA of Homo sapiensis approximately 59%, as compared to the percentage cytosine plusguanine (C+G). The adenine plus thymine content in P. falciparum isapproximately 80%. The adenine plus thymine content in the CS gene of P.falciparum is approximately 87%. To obtain sufficient protection, it isbelieved to be necessary to improve production levels in the host. Oneway to achieve this is to optimize codon usage by altering the nucleicacid content of the antigenic determinant in the viral-based vector,without altering the amino acid molecule thereof. For this, thereplication-defective recombinant viral vectors as described herein havean adenine plus thymine content in the heterologous nucleic acids of theinvention of less than 87%, preferably less than 80%, and morepreferably less than or equal to approximately 59%. Based on codon usagein humans and the amino acid content of the CS genes of P. falciparumand yoelii, the percentages of the codon-optimized genes were evenlower, reaching approximately 45% for the amino acid content asdisclosed herein. Therefore, as far as the CS genes are concerned, it ispreferred to have an adenine plus thymine content of approximately 45%.It is to be understood that if a species other than humans is to betreated, which may have a different adenine plus thymine concentration(less or more than 59%), and/or a different codon usage, that the genesencoding the CS proteins of the invention may be adjusted to fit therequired content and give rise to suitable expression levels for thatparticular host. Of course, it cannot be excluded either that slightchanges in content may result in slight expression level changes indifferent geographical areas around the world. It is also to beunderstood that with slight changes in the number of repeats included inthe amino acid molecule of the proteins, that percentages may differaccordingly. All these adjusted contents are part hereof.

The invention is further described with the aid of the followingillustrative Examples.

EXAMPLES Example 1 Assembly of the Plasmodium falciparumCircumsporozoite Synthetic Gene

Comparative studies conducted with DNA vaccines based on native andcodon-optimized genes encoding merozoite proteins of P. falciparum haveindicated a direct correlation with expression levels and immunogenicity(Narum et al. 2001). A new sequence of the gene encoding the Plasmodiumfalciparum circumsporozoite (CS) protein was designed. Studies onpopulations of malaria parasites obtained from widely separatedgeographical regions have revealed the presence of CS sequencepolymorphism. The new P. falciparum CS sequence was assembled byalignment of the different available protein sequences present in theGeneBank database (listed in Table I). First, all the differentsequences were placed in order of subgroups based on global location orby lab strain where the isolates originated. All CS complete or partialsequences were used in order to identify variation between the differentgeographical areas and identified lab strains. The final amino acidconsensus sequence determined was thoroughly examined. The inventors ofthe invention subsequently adjusted this consensus sequence to have anew CS gene synthesized (FIG. 1). The novel amino acid molecule is shownin FIG. 1A. The new CS protein harbors the aspects listed below (fromN-terminus to C-terminus):

The N-terminal signal sequence, which would direct the protein to theendoplasmic reticulum, is left unchanged.

The HLA-binding peptide amino acid (31-40), as well as region 1(predominant B-cell epitope) are conserved; therefore, these sequencesare left unchanged.

A number of repeats: there are 14-41 NANP (SEQ ID NO:10) repeats in thedifferent isolates and 4 NVDP (SEQ ID NO:11) repeats. It was chosen toincorporate 27 NANP repeats, a cluster of 3 NVDP repeats, and oneseparate NVDP repeat.

The ENANANNAVKN (SEQ ID NO:12) sequence directly downstream of therepeats mentioned above was found to be reasonably conserved betweenstrains.

The Th2R region and the immunodominant CD8 epitope (Lockyer et al. 1989;Zevering et al. 1994): a single consensus sequence that differs in somerespects from that of the known and frequently used lab strain 3D7sequence was determined. This sequence is sometimes referred to as the“universal epitope” in literature (Nardin et al. 2001).

The region 2, overlapping with the Th2R region, remained conserved.

The TH3R region, which is considered to be a less important CD8 epitope,is used in the form of a consensus sequence, since only point mutationswere found.

The C-terminal 28 amino acids, which constitute a GPI signal anchorsequence that is inefficient in mammalian cells (Moran and Caras, 1994)and not hydrophobic by itself, serve as a stable membrane anchor. Thegene was constructed such that the whole sequence can be removed, butalso leaving open the possibility of remaining present. This allows acomparison of the antigenicity of adenovirus vectors carrying afull-length CS versus those expressing the protein deleted in the GPIsignal anchor sequence. In fact, it has been described that removal ofthe GPI signal sequence from a CS DNA vaccine enhanced induction onimmune response against malaria infection in rodents (Scheiblhofer etal. 2001).

Substitution S to A at position 373: this amino acid substitution wasintroduced to eliminate a potential glycosylation site recognized bymammalian cells.

Since the malaria parasite residue usage (87% A and T) is significantlydifferent from that of the Homo sapiens, the gene encoding the newlydesigned CS protein was codon optimized in order to improve itsexpression in mammalian cells, taking care of the following aspects toavoid cis-acting sequences: no premature poly(A) sites and internal TATAboxes should be present; Chi-sites, ribosomal entry sites and AT-richsequence clusters should be avoided; no (cryptic) splice-acceptor and-donor sites should be present; repetitive sequence stretches should beavoided as much as possible; and GC-rich sequences should also beavoided. The final codon-optimized gene is shown in FIG. 1B.

The newly designed CS consensus sequence was synthesized and cloned intopCR-script (Stratagene) by GeneArt (Regensburg, Germany), usingmethodology known to persons skilled in the art of synthetic DNAgeneration, giving rise to a clone named 02-148 (pCR-script.Pf) (SEQ IDNO:1).

Next to this synthetic clone, another synthetic gene was generated,wherein a number of mutations were introduced in the 3′ end to obtain anamino acid molecule that is identical to the P. falciparum CS protein ofthe 3D7 strain, which is deleted in the last 14 amino acids (FIG. 2).This gene was also codon optimized using the same provisions asdescribed above and subsequently synthesized and cloned into pCR-script(Stratagene) by GeneArt. The clone was named 02-659 (pf-aa-sub) (SEQ IDNO:4).

Example 2 Codon Optimization of the Circumsporozoite Gene of theRodent-Specific Malaria Parasite Plasmodium yoelii

Malaria species that have been adapted to robust rodent models, such asP. berghei and P. yoelii, have been powerful tools for identificationand testing of malaria candidate vaccines. Since infectivity of P.yoelii sporozoites resembles that of P. falciparum, it was decided tomake use of the P. yoelii model for exemplification of the capability ofAd35 vectors carrying codon-optimized CS proteins to provide sterileimmunity and, therefore, protection against malaria infection. The P.yoelii CS gene, encoding for residues 1-356 as previously described(Rodrigues et al. 1997), was codon optimized using the same provisionsas described above and synthesized by GeneArt (GmbH-Regensburg,Germany). The sequence of the codon-optimized P. yoelii CS gene (plasmid02-149) is depicted in FIG. 3.

Example 3 Generation of Recombinant Adenoviral Vectors Based on Ad5

RCA-free recombinant adenoviruses can be generated very efficientlyusing adapter plasmids, such as pAdApt, and adenovirus plasmidbackbones, such as pWE/Ad.AflII-rITRsp. Methods and tools have beendescribed extensively elsewhere (WO 97/00326, WO 99/55132, WO 99/64582,WO 00/70071, and WO 00/03029). Generally, the adapter plasmid containingthe transgene of interest in the desired expression cassette is digestedwith suitable enzymes to free the recombinant adenovirus sequences fromthe plasmid vector backbone. Similarly, the adenoviral complementationplasmid pWE/Ad.AflII-rITRsp is digested with suitable enzymes to freethe adenovirus sequences from the vector plasmid DNA.

The cloning of the gene encoding the CS protein from P. yoelii parasiteinto pIPspAdapt1 was performed as follows. Plasmid 02-149 (GeneArt, seeabove) containing the codon-optimized CS gene was digested with HindIIIand BamHI restriction enzymes. The 1.1 Kb fragment corresponding to theP. yoelii CS gene was isolated from agarose gel and ligated to HindIIIand BamHI-digested pIPspAdapt1 vector (described in WO 99/64582). Theresulting plasmid was named pAdapt.CS.Pyoel and contains the CS geneunder the transcriptional control of full-length human immediate-early(1E) cytomegalovirus (CMV) promoter and a downstream SV40 poly(A)signal.

The cloning of the gene encoding the full-length CS protein from P.falciparum parasite into pIPspAdapt1 was performed as follows. Plasmid02-148 pCR-script.Pf (see above) containing the codon-optimized CS genewas digested with HindIII and BamHI restriction enzymes. The 1.2 kbfragment corresponding to the CS gene was isolated from agarose gel andligated to HindIII and BamHI-digested pIPspAdapt1 vector. The resultingplasmid was named pAdapt.CS.Pfalc and contains the CS gene under thetranscriptional control of full-length human immediate-early (1E)cytomegalovirus (CMV) promoter and the downstream SV40 poly(A) signal.

The cloning of the gene encoding the CS P. falciparum protein minus theGPI anchor sequence, thus with the deletion of the last 28 amino acids,into pIPspAdapt1 was performed as follows. A 1.1 kb PCR fragment wasamplified using plasmid 02-148 as template, with the primers Forw.Falc(5′-CCA AGC TTG CCA CCA TGA TGA GG-3′) (SEQ ID NO:13) and Rev.Falc.CS-28 (5′-CCG GAT CCT CAG CAG ATC TTC TTC TCG-3′) (SEQ ID NO:14).Primers were synthesized by Invitrogen. For the PCR, the enzyme Pwo DNApolymerase (Inno-train Diagnostic) was used, while the following programwas applied: one cycle of 5 minutes at 94° C., 1 minute at 50° C., 2minutes 30 seconds at 72° C.; five cycles of 1 minute at 94° C., 1minute at 50° C., 2 minutes 50 seconds at 72° C.; twenty cycles of 1minute at 94° C., 1 minute at 54° C., 2 minutes 50 seconds at 72° C.;and one cycle of 1 minute at 94° C., 1 minute at 54° C., followed by 10minutes at 72° C. The amplified PCR product was digested with therestriction enzymes HindIII and BamHI and then cloned into pIPspAdapt1which was also digested with HindIII and BamHI. The resulting plasmidwas designated pAdapt.CS.Pfalc(−28) and contains the CS gene under thetranscriptional control of full-length human immediate-early (1E)cytomegalovirus (CMV) promoter and the downstream SV40 poly(A) signal.

The cloning of the gene encoding the CS P. falciparum protein minus theGPI anchor, thus with the deletion of the last 14 amino acids, intopIPspAdapt1 was performed as follows. A 1.1 kb PCR fragment wasamplified using plasmid 02-148 as template, with the primers Forw.Falc(SEQ ID NO:13) and Rev.Falc. CS-14 (5′-CCG GAT CCT CAG CTG TIC ACC ACGTTG-3′) (SEQ ID NO:15). Primers were synthesized by Invitrogen. For thePCR, the enzyme Pwo DNA polymerase (Inno-train Diagnostic) was used,while the following program was applied: one cycle of 5 minutes at 94°C., 1 minute at 50° C., 2 minutes 30 seconds at 72° C.; five cycles of 1minute at 94° C., 1 minute at 50° C., 2 minutes 50 seconds at 72° C.;twenty cycles of 1 minute at 94° C., 1 minute at 54° C., 2 minutes 50seconds at 72° C.; and one cycle of 1 minute at 94° C., 1 minute at 54°C., followed by 10 minutes at 72° C. The amplified PCR product wasdigested with the restriction enzymes HindIII and BamHI and then clonedinto pIPspAdapt1 also digested with HindIII and BamHI. The resultingplasmid was designated pAdapt.CS.Pfalc(−14) and contains the CS geneunder the transcriptional control of full-length human immediate-early(1E) cytomegalovirus (CMV) promoter and the downstream SV40 poly(A)signal.

The cloning of the gene encoding the CS P. falciparum protein minus theGPI anchor, displaying a C-terminal sequence as in the 3D7 strain, intopIPspAdapt1 is performed as follows. Plasmid 02-659 pf-aa-sub (seeabove) containing the codon-optimized CS gene is digested with HindIIIand BamHI restriction enzymes. The 1.1 kb fragment corresponding to theCS gene is ligated to HindIII and BamHI-digested pIPspAdapt1 vector. Theresulting plasmid is designated pAdapt.CS.Pfalc (pf-aa-sub) and containsthe CS gene under the transcriptional control of full-length humanimmediate-early (1E) cytomegalovirus (CMV) promoter and the downstreamSV40 poly(A) signal.

The generation of the recombinant virus named Ad5ΔE3.CS.Pyoel wasperformed as follows. pAdapt.CS.Pyoel was digested by PacI restrictionenzyme to release the left-end portion of the Ad genome. PlasmidpWE/Ad.AflII-rITRsp containing the remaining right-end part of the Adgenome has a deletion of 1878 bp in the E3 region (XbaI deletion). Thisconstruct was also digested with PacI. pAdapt.CS.Pyoel was separatelytransfected with PacI-digested pWE.Ad.AflII-rITRsp into PER-E1B55Kproducer cells (cells have been described in WO 02/40665) usinglipofectamine transfection reagent (Invitrogen) using methods known inthe art and as described in WO 00/70071. Homologous recombinationbetween overlapping sequences led to generation of the recombinant virusnamed Ad5ΔE3.CS.Pyoel. It is to be understood that Ad5-based vectors canalso be produced on the aforementioned PER.C6® producer cells, whichcells are represented by the cells deposited under ECACC no. 96022940(see above). The adenoviral vector, in crude lysates, resulting fromthis transfection was plaque purified using methods known to personsskilled in the art. Single plaques were analyzed for the presence of theCS transgene and amplified for large-scale production in triple-layerflasks (3×175 cm²/flask). Upon amplification, cells are harvested atfull CPE and the virus is purified by a two-step Cesium Chloride (CsCl)purification procedure as routinely done by those skilled in the art andgenerally as described in WO 02/40665.

The generation of the recombinant virus named Ad5ΔE3.CS.Pfalc wasperformed as follows. pAdapt.CS.Pfalc was digested by PacI restrictionenzyme to release the left-end portion of the Ad genome. PlasmidpWE/Ad.AflII-rITRsp containing the remaining right-end part of the Adgenome has a deletion of 1878 bp in the E3 region (XbaI deletion). Thisconstruct was also digested with PacI. pAdapt.CS.Pfalc was transfectedwith PacI-digested pWE.Ad.AflII-rITRsp into PER-E1B55K producer cellsusing lipofectamine transfection reagent. Homologous recombinationbetween overlapping sequences led to generation of the recombinant virusnamed Ad5ΔE3.CS.Pfalc. The adenoviral vector in crude lysates resultingfrom this transfection was plaque purified using methods known topersons skilled in the art. Single plaques were analyzed for thepresence of the CS transgene and amplified for large-scale production intriple-layer flasks (3×175 cm²/flask). Cells were harvested at full CPEand the virus was purified by a two-step CsCl purification procedure asroutinely done by those skilled in the art and generally as described inWO 02/40665.

The generation of the recombinant viruses named Ad5ΔE3.CS.Pfalc(−28) andAd5ΔE3.CS.Pfalc(−14) was performed as follows. pAdapt.CS.Pfalc(−28) andpAdapt.CS.Pfalc(−14) were separately digested by PacI restriction enzymeto release the left-end portion of the Ad genome. PlasmidpWE/Ad.AflII-rITRsp containing the remaining right-end part of the Adgenome has a deletion of 1878 bp in the E3 region (XbaI deletion). Thisconstruct was also digested with PacI. pAdapt.CS.Pfalc(−28) andpAdapt.CS.Pfalc(−14) were separately transfected with PacI-digestedpWE.Ad.AflII-rITRsp into PER-E1B55K producer cells using lipofectaminetransfection reagent. Homologous recombination between overlappingsequences led to generation of recombinant viruses named, respectively,Ad5ΔE3.CS.Pfalc(−28) and Ad5ΔE3.CS.Pfalc(−14). Adenoviral vectors incrude lysates resulting from these transfections are plaque purifiedusing methods known to persons skilled in the art. Single plaques areanalyzed for the presence of the CS transgene and amplified forlarge-scale production in triple-layer flasks (3×175 cm²/flask). Cellsare harvested at full CPE and the virus is purified by a two-step CsClpurification procedure as routinely done by those skilled in the art andgenerally as described in WO 02/40665.

The generation of the recombinant virus named Ad5ΔE3.CS.Pfalc(pf-aa-sub)is performed as follows. pAdapt.CS.Pfalc(pf-aa-sub) is digested by PacIrestriction enzyme to release the left-end portion of the Ad genome.Plasmid pWE/Ad.AflII-rITRsp containing the remaining right-end part ofthe Ad genome is also digested with PacI. pAdapt.CS.Pfalc(pf-aa-sub) istransfected with PacI-digested pWE.Ad.AfflII-rITRspΔE3 into PER.C6® orPER-E1B55K producer cells using lipofectamine transfection reagent, orby other means such as electroporation or other transfection methodsknown to persons skilled in the art. Homologous recombination betweenoverlapping sequences leads to generation of the recombinant virus namedAd5ΔE3.CS.Pfalc (pf-aa-sub). The adenoviral vector, in crude lysates,resulting from this transfection is plaque purified using methods knownto persons skilled in the art. Single plaques are analyzed for thepresence of the CS transgene and amplified for large-scale production intriple-layer flasks (3×175 cm²/flask). Cells are harvested at full CPEand the virus is purified by a two-step CsCl purification procedure asroutinely done by those skilled in the art and generally as described inWO 02/40665.

Next to these procedures, generation of the control recombinantadenovirus named Ad5ΔE3.empty was carried out as described above, usingas adapter, the plasmid pAdapt, lacking a transgene.

Example 4 Generation of Recombinant Adenoviral Vaccine Vectors Based onAd35

A first 101 bp PCR fragment containing the Ad5 pIX promoter (nucleotides1509-1610) was generated with the primers SV40for (5′-CAA TGT ATC TTATCA TGT CTA G-3′) (SEQ ID NO:16) and pIX5Rmfe (5′-CTC TCT CAA TTG CAGATA CAA AAC TAC ATA AGA CC-3′) (SEQ ID NO:17). The reaction was donewith Pwo DNA polymerase according to the manufacturer's instructions butwith 3% DMSO in the final mix. pAdApt was used as a template. Theprogram was set as follows: 2 minutes at 94° C.; thirty cycles of: 30seconds at 94° C., 30 seconds at 52° C. and 30 seconds at 72° C.;followed by 8 minutes at 72° C. The resulting PCR fragment contains the3′ end of the SV40 poly(A) signal from pAdApt and the Ad5-pIX promotorregion as present in Genbank Accession number M73260 from nucleotide3511 to nucleotide 3586 and an MfeI site at the 3′ end. A second PCRfragment was generated as described above but with primers pIX35Fmfe(5′-CTC TCT CAA TTG TCT GTC TTG CAG CTG TCA TG-3′) (SEQ ID NO:18) and35R4 (for reference to the sequence of the 35R4 primer, see WO00/70071). pAdApt35IP1 (described in WO 00/70071) was used as atemplate, the annealing was set at 58° C. for 30 seconds and theelongation of the PCR program was set at 72° C. for 90 seconds. This PCRprocedure amplifies Ad35 sequences from nucleotide 3467 to nucleotide4669 (sequence numbering as in WO 00/70071) and adds an MfeI site to the5′ end. Both PCR fragments were digested with MfeI and purified usingthe Qiagen PCR purification kit (Qiagen). Approximate equimolar amountsof the two fragments were used in a ligation reaction. Following anincubation of two hours with ligase enzyme in the correct buffers atroom temperature, the mixture was loaded on an agarose gel and the DNAfragments of 1.4 kb length were isolated with the Geneclean II kit(BIO101, Inc). The purified DNA was used in a PCR amplification reactionwith primers SV40for and 35R4. The PCR was done as described above withan annealing temperature of 52° C. and an elongation time of 90 seconds.The resulting product was isolated from gel using the Qiagen gelextraction kit and digested with AgeI and BglII. The resulting 0.86 kbfragment containing the complete 100 nucleotide pIX promoter form Ad5,the MfeI site and the pIX ORF (fragment MfeI-AgeI, including the ATGstart site) from Ad35, but without a poly(A) sequence, was isolated fromgel using the Geneclean II kit.

RCA-free recombinant adenoviruses based on Ad35 can be generated veryefficiently using adapter plasmids, such as pAdApt535 (described below)and adenovirus plasmid backbones, such as pWE/Ad35.pIX-rITRΔE3(described in WO 02/40665). To generate pAdApt535, pAdApt35.Luc(described in WO 00/70071) was digested with BglII and AgeI and theresulting 5.8 kb vector was isolated from gel. This fragment was ligatedwith the isolated 0.86 kb BglII-AgeI fragment containing the Ad5-Ad35chimeric pIX promotor described above, to result in a plasmid namedpAdApt535.Luc, which was subsequently digested with BglII and ApaI. Theresulting 1.2 kb insert was purified over gel. pAdApt35IP1 was digestedwith BglII and ApaI and the 3.6-kb vector fragment was isolated overgel. Ligation of the 1.2 kb BglII-ApaI insert from pAdApt535.Luc and the3.6 kb BglII-ApaI-digested vector resulted in pAdApt535.

The cloning of the gene encoding the CS protein from P. yoelii parasiteinto pAdapt535 was performed as follows. Plasmid 02-149 containing thecodon-optimized P. yoelii CS gene (see above) was digested with therestriction enzymes HindIII and BamHI. The 1.1 kb fragment correspondingto the CS gene was isolated over agarose gel and ligated to the HindIIIand BamHI-digested pAdapt535 vector. The resulting plasmid was namedpAdapt535-CS.Pyoel and contains the CS gene under the transcriptionalcontrol of the full-length human CMV promoter and the downstream SV40poly(A) signal.

The cloning of the gene encoding the full-length CS protein from P.falciparum parasite into pAdapt535 was performed as follows. Plasmid02-148 (pCR-script.Pf) containing the codon-optimized CS gene of P.falciparum was digested with the restriction enzymes HindIII and BamHI.The 1.2 kb fragment corresponding to the CS gene was isolated overagarose gel and ligated to the HindIII and BamHI-digested pAdapt535vector. The resulting plasmid was named pAdapt535-CS.Pfalc and containsthe CS gene under the transcriptional control of the full-length humanCMV promoter and the downstream SV40 poly(A) signal.

The cloning of the gene encoding the CS P. falciparum protein minus theGPI anchor sequence, thus with the deletion of the last 28 amino acids,into pAdapt535 is performed as follows. The 1.1 kb PCR fragment obtainedas described above using primers Forw.Falc and Rev. Falc.CS-28 isdigested with the restriction enzymes HindIII and BamHI and then clonedinto pAdapt535 vector also digested with HindIII and BamHI. Theresulting plasmid is designated pAdapt535.CS.Pfalc(−28) and contains theCS gene under the transcriptional control of the full-length human CMVpromoter and the downstream SV40 poly(A) signal.

The cloning of the gene encoding the CS P. falciparum protein minus theGPI anchor sequence, now with the deletion of the last 14 amino acids,into pAdapt535 is performed as follows. The 1.1 kb PCR fragment obtainedas described above using primers Forw.Falc and Rev. Falc.CS-14 isdigested with the restriction enzymes HindIII and BamHI and then clonedinto pAdapt535 vector also digested with HindIII and BamHI. Theresulting plasmid is designated pAdapt535.CS.Pfalc(−14) and contains theCS gene under the transcriptional control of the full-length human CMVpromoter and the downstream SV40 poly(A) signal.

The cloning of the gene encoding the CS P. falciparum protein minus theGPI anchor sequence, displaying a C-terminus sequence as in the 3D7strain, into pAdapt535 is performed as follows. Plasmid 02-659 pf-aa-sub(see above) containing the codon-optimized CS gene is digested withHindIII and BamHI restriction enzymes. The 1.1 kb fragment correspondingto the CS gene is ligated to HindIII and BamHI-digested pAdapt535vector. The resulting plasmid is designatedpAdapt535.CS.Pfalc(pf-aa-sub) and contains the CS gene under thetranscriptional control of the full-length human CMV promoter and thedownstream SV40 poly(A) signal.

The generation of the recombinant virus named Ad35ΔE3.CS.Pyoel wasperformed as follows. pAdapt535.CS.Pyoel was digested by PacIrestriction enzyme to release the left-end portion of the Ad genome.Plasmid pWE.Ad35.pIX-rITRΔE3 containing the remaining right-end part ofthe Ad genome with a deletion of 2673 bp in the E3 region is digestedwith NotI. pAdapt535.CS.Pyoel was transfected with NotI-digestedpWE.Ad35.pIX-rITRΔE3 into PER-E1B55K producer cells using lipofectaminetransfection reagent. The generation of the cell line PER-E1B55K hasbeen described in detail in WO 02/40665. In short, this publicationdescribes that PER.C6® cells were stably transfected with ScaIlinearized pIG35-55K DNA, carrying the E1B-55K gene of adenovirusserotype 35, after which a selection procedure with G418 yielded in 196picked colonies. Further culturing of a limited number of well-growingcolonies resulted in stable cell lines that, upon numerous subcultures,stably expressed the Ad35 E1B-55K gene and supported the growth ofrecombinant Ad35 viruses, while the original PER.C6® cell were veryinefficient in supporting this.

Homologous recombination between overlapping sequences led to generationof the recombinant virus named Ad35ΔE3.CS.Pyoel. The adenoviral vector,in crude lysates, resulting from this transfection was plaque purifiedusing methods known to persons skilled in the art. Single plaques wereanalyzed for the presence of the CS transgene and amplified forlarge-scale production in triple-layer flasks (3×175 cm²/flask). Uponamplification, cells were harvested at full CPE and the virus waspurified by a two-step CsCl purification procedure as routinely done bythose skilled in the art and generally as described in WO 02/40665.

The generation of the recombinant virus named Ad35ΔE3.CS.Pfalc wasperformed as follows. pAdapt535.CS.Pfalc was digested by PacIrestriction enzyme to release the left-end portion of the Ad genome.Plasmid pWE.Ad35.pIX-rITRΔE3 containing the remaining right-end part ofthe Ad genome with a deletion of 2673 bp in the E3 region was digestedwith NotI. pAdapt535.CS.Pfalc was transfected with NotI-digestedpWE.Ad35.pIX-rITRΔE3 into PER-E1B55K producer cells using lipofectaminetransfection reagent. Homologous recombination between overlappingsequences led to generation of the recombinant virus namedAd35ΔE3.CS.Pfalc. The adenoviral vector in crude lysates resulting fromthis transfection was plaque purified using methods known to personsskilled in the art. Single plaques were analyzed for the presence of theCS transgene and amplified for large-scale production in triple-layerflasks (3×175 cm²/flask). Upon amplification, cells were harvested atfull CPE and the virus was purified by a two-step CsCl purificationprocedure as routinely done by those skilled in the art and generally asdescribed in WO 02/40665.

The generation of the recombinant viruses named Ad35ΔE3.CS.Pfalc(−28)and Ad35ΔE3.CS.Pfalc(−14) is performed as follows.pAdapt535.CS.Pfalc(−28) and pAdapt535.CS.Pfalc(−14) were separatelydigested by PacI restriction enzyme to release the left-end portion ofthe Ad genome. Plasmid pWE.Ad35.pIX-rITRΔE3 containing the remainingright-end part of the Ad genome is digested with NotI.pAdapt535.CS.Pfalc(−28) and pAdapt535.CS.Pfalc(−14) are separatelytransfected with NotI-digested pWE.Ad35.pIX-rITRΔE3 into PER-E1B55Kproducer. Homologous recombination between overlapping sequences leadsto generation of recombinant viruses named, respectively,Ad35ΔE3.CS.Pfalc(−28) and Ad35ΔE3.CS.Pfalc(−14). Adenoviral vectors incrude lysates resulting from these transfections are plaque purifiedusing methods known to persons skilled in the art. Single plaques areanalyzed for the presence of the CS transgene and amplified forlarge-scale production in triple-layer flasks (3×175 cm²/flask). Cellsare harvested at full CPE and the virus is purified by a two-step CsClpurification procedure as routinely done by those skilled in the art andgenerally as described in WO 02/40665.

The generation of the recombinant virus namedAd35ΔE3.CS.Pfalc(pf-aa-sub) is performed as follows.pAdapt535.CS.Pfalc(pf-aa-sub) is digested by PacI restriction enzyme torelease the left-end portion of the Ad genome. PlasmidpWE.Ad35.pIX-rITRΔE3 containing the remaining right-end part of the Adgenome is digested with NotI. pAdapt535.CS.Pfalc(pf-aa-sub) istransfected with NotI-digested pWE.Ad35.pIX-rITRΔE3 into PER-E1B55Kproducer cells using lipofectamine transfection reagent (Invitrogen)using methods known in the art and as described in WO 00/70071 or byelectroporation or other transfection methods known to those skilled inthe art. Homologous recombination between overlapping sequences leads togeneration of the recombinant virus named Ad35ΔE3.CS.Pfalc(pf-aa-sub).The adenoviral vector in crude lysates resulting from this transfectionis plaque purified using methods known to persons skilled in the art.Single plaques are analyzed for the presence of the CS transgene andamplified for large-scale production in triple-layer flasks (3×175cm²/flask). Cells are harvested at full CPE and the virus is purified bya two-step CsCl purification procedure as routinely done by thoseskilled in the art and generally as described in WO 02/40665.

Example 5 Inducing Protection Against P. yoelii Malaria Infection UsingRecombinant Adenoviral-Based Vaccines In Vivo

Ad5-based vectors genetically engineered to express the CS antigen ofthe rodent malaria P. yoelii have been shown capable to induce completeprotection against P. yoelii infection (Rodrigues et al. 1997). Aside-by-side comparison between Ad5 and Ad35 vectors carrying thecodon-optimized P. yoelii CS gene was designed to investigate the immuneresponse that is induced and to investigate their ability in raisingprotection against P. yoelii parasite infection in mice. The studyenrolled Balb/C mice that were immunized by intra-muscular orsubcutaneous injection of 10⁸-10¹⁰ viral particles (vp) of Ad5ΔE3- orAd35ΔE3-based viral vectors (as described above) carrying either the P.yoelii CS gene (Ad5ΔE3-CS.Pyoel and Ad35ΔE3-CS.Pyoel) or no transgene(Ad5ΔE3-empty and Ad35ΔE3-empty). FIG. 4 shows the results of theexperiments wherein the administration route was compared using bothvectors. The number of IFN-γ-secreting cells in a population of 10⁶splenocytes was determined (FIG. 4A), as well as the antibody titers inthe serum (FIG. 4B). The experiments were performed on mice that weresacrificed two weeks after injection with the recombinant adenoviruses.Each of the bars represents the average of five mice. If mice were notsacrificed, they were used for a challenge with live sporozoites, afterwhich the rate of protection was determined (FIGS. 5A and B). Each ofthese bars represents the average of five mice. The experiments onhumoral and cellular immune responses are performed with immunologicalassays well known to persons skilled in the art and as described, forinstance, by Bruña-Romero et al. (2001a). The immunization, challengeand read out are scheduled in Tables II and III. Antibodies titersagainst sporozoites can be determined by an indirect immunofluorescenceassay or with an ELISA. FIG. 4B shows the results as calculated with anELISA. Cellular immune responses were determined by ex vivo ELISPOTassay measuring the relative number of CS-specific, IFN-γ-secreting,CD8+ and CD4+ T-cells. Protection against malaria infection wasmonitored by determining the levels of parasite inhibition in the liversof immunized mice through reverse transcriptase PCR quantification of P.yoelii ribosomal RNA copies.

The immunization with Ad5- and Ad35-based vectors was performed asfollows. Aliquots of recombinant adenoviruses that were stored at −70°C. were gradually thawed on ice and diluted to 100 μl in the desiredconcentration in PBS with 1% heat-inactivated Normal Mouse Serum.Subsequently, the samples were sonicated for five seconds. Subcutaneousadministration was performed at both sides of the tail base with avolume of 50 μl at each side. Intra muscular administration wasperformed in both thighs with a volume of 50 μl at each thigh.

The Indirect Immunofluorescence Assay (“IFA”) is performed according toBruña-Romero et al. (2001a). First, infected mosquitoes are generated byinitially having a native mouse infected with an infected mosquito byhaving the mouse bitten at three different sites. Blood is removed fromthe mouse after eight days when parasitemia is 4-8% and diluted to 1%.Then, other naïve mice are injected i.p. with the diluted blood sample.After three days, the blood is taken which serves as a blood meal forstarved mosquitoes. These are fed for two days. After 14 days, P. yoeliisporozoites are isolated from the blood-fed mosquitoes by anesthetizinginfected mosquitoes on ice and subsequently saturating them in 70%ethanol. Then, the mosquitoes are transferred to PBS pH 7.4 and thesalivary glands are dissected. These are subsequently grinded on ice andthe sporozoites are separated from the debris by centrifugation. Usingthis method, approximately 35,000 P. yoelii sporozoites can be obtainedfrom one mosquito. Then, glass slides in a 12-multi-well plate arecoated with approximately 10,000 P. yoelii sporozoites, each inDulbecco's Modified Eagle's Medium (DMEM) plus 10% Fetal Bovine Serum byair drying. A range of dilutions of sera of the vaccinated mice (in avolume of 10 μl in PBS plus 5% FBS) is subsequently incubated with theair-dried sporozoites for 30 minutes at room temperature in a moisturesenvironment. Then, the slides are aspirated, washed twice with PBS and10 μl of a 30-fold diluted FITC-conjugated Goat-anti-Mouse antibody(Kirkegaard & Perry Laboratories, USA, catalogue no. 02-18-06) is addedand incubated for 30 minutes at room temperature. Wells were againaspirated and washed twice. For counter-staining, a solution of 100μg/ml Ethidium Bromide is incubated for ten minutes, after which theaspiration step is repeated and the wells are washed with water. Slidesare mounted using permount containing phenylenediamine/anti-fade. Theanti-sporozoite antibody titers are determined as the highest serumdilution-producing fluorescence. For the determination of antibodytiters, one can also use an ELISA. For this, ELISA plates (Immulon II,Dynatech) were coated with 2 μg/ml antigen in PBS by adding 100 μl perwell of this solution and leaving it overnight at 4° C. The antigen thatwas used is a 3×6 amino acid repeat of the P. yoelii CS protein:QGPGAPQGPGAPQGPGAP (SEQ ID NO:19). The plates were subsequently washedthree times with washing buffer (1×PBS, 0.05% TWEEN™), and 200 μlblocking buffer (10% FCS in washing solution) was added per well. Plateswere incubated for one to two hours at room temperature. Then, plateswere washed three times again with washing buffer including 5% FCS.Dilutions of the sera were made as follows: 50 μl washing buffer plus 5%FCS was added to wells 2-12. Then 100 μl washing buffer plus 5% FCS isadded to the first well and 1:2 serial dilutions are made bytransferring 50 μl from well 1 to 2, then from 2 to 3, etc. Plates areincubated for one hour at room temperature. Then, the plates are washedthree times with washing buffer and 100 μl of a 1:2000 dilutedperoxidase-labeled Goat anti-Mouse IgG (anti Heavy and Light chain,human absorbed, Kirkegaard & Perry Laboratories, catalogue no. 074-1806)is added per well and incubated. Then, plates are washed with washingbuffer three times and once with PBS and then 100 μl ABTS substratesolution (ABTS 1-Component, Kirkegaard & Perry Laboratories, cataloguenumber 50-66-18) is added to each well. The reaction is terminated bythe addition of 50 μl 1% SDS and plates are read at 405 nm in an ELISAreader.

The ELISPOT assay to determine the relative number of CS-specificIFN-γ-secreting CD8+ and CD4+ T-cells in the spleen, and the reversetranscriptase PCR and real-time PCR to quantify the amount ofparasite-specific RNA present in the liver of the challenged mice wereall performed as described by Bruña-Romero et al. (2001a and 2001b),except for the fact that the number of cycles in the real-time PCR was45.

While attenuation P. yoelii infection in Ad5ΔE3-CS.Pyoel vaccinerecipients is predicted (Rodrigues et al. 1997), vaccination withAd35ΔE3-CS.Pyoel is expected to be superior or at least equallyeffective.

FIG. 4A shows that with an administration of 10⁹ and 10¹⁰ viralparticles per mouse, the Ad35-based vector is at least as effective ininducing a cellular immune response as the Ad5-based vector, if notsuperior. It can be concluded that with this set-up, there is nodramatic difference in cellular response as indicated by the number ofIFN-γ-secreting cells after intramuscular and subcutaneous delivery.

FIG. 4B shows the antibody titers in the same experiment and performedon the same sera using the indirect immuno-fluorescence experimentoutlined above. If compared to the results shown in FIG. 4A, it is clearthat at a dose of 10⁹ viral particles, the Ad35-based vector induces asignificant cellular immune response but does not give rise to very hightiters of anti-sporozoite antibodies. Again, there is not a significantdifference between the two routes of administration.

Animals that received different doses of Ad5- and Ad35-based vectorsexpressing the codon-optimized P. yoelii CS antigen, were subsequentlychallenged i.v. with 10⁵ sporozoites purified as described above. Theresults of these experiments are shown in FIGS. 5A and B. The percentageof inhibition was calculated as compared to naïve mice that were notimmunized.

Mice that were immunized received s.c. 10⁹ or 10¹° viral particles (vp)and were challenged after 14 days with the sporozoites and thensacrificed after 48 hours. Negative controls were empty vectors withoutantigen and non-immunized mice. Clearly, a high percentage of inhibitionis obtained when using the Ad5-based vector as well as with theAd35-based vector, applying the two doses, while no protection was foundin the negative controls (FIG. 5A). Importantly, only a low number ofparasite-specific 18S ribosomal RNAs could be determined in the liver ofthe immunized mice, while the mice that received no adenoviral vector orempty vectors contained large numbers of these RNAs (FIG. 5B). Thisstrongly indicates that the Ad35-based vector, like the Ad5-basedvector, can give rise to significant protection against the malariaparasite, even after a single round of immunization.

Example 6 Inducing Immunity Against P. falciparum Malaria InfectionUsing Recombinant Adenoviral-Based Vaccines In Vivo

A side-by-side comparison between Adenovirus serotype 5 (Ad5) andAdenovirus serotype 35 (Ad35) vectors is designed to investigate theability to induce humoral and cellular immune responses against the CSantigen of the P. falciparum parasite in mice. In addition,immunogenicities of Adenovirus vectors containing full-length and GPIminus CS are compared. This study enrolls B10.BR mice. Animals areimmunized by intramuscular injection of 10⁸-10¹⁰ vp of Ad5ΔE3 or Ad35ΔE3viral vectors carrying either the full-length CS gene (Ad5ΔE3-CS.Pfalcand Ad35ΔE3-CS.Pfalc) or the GPI-anchor sequence minus CS gene(Ad5ΔE3-CS.Pfalc.(−28)/(−14) and Ad35ΔE3-CS.Pfalc.(−28)/(−14)) or notransgene (Ad5ΔE3-empty and Ad35ΔE3-empty). At two weeks and six toeight weeks post-vaccination, cellular and humoral responses aremonitored with immunological assays well known to persons skilled in theart as described above. The immunization, challenge and read out arescheduled in Tables IV and V.

Immunogenicity of the Ad35-based vectors is expected to be superior orat least comparable to the immunogenicity triggered by Ad5-basedvectors. FIG. 6 shows the results that were obtained by using theAd5-based vector containing the full-length gene encoding the P.falciparum CS protein, the gene encoding the protein with the 14 aminoacid deletion and the gene encoding the protein with the 28 amino aciddeletion. The results indicate that all three (Ad5-based) vectors areable to induce a cellular immune response as measured by the number ofCS-specific IFN-γ-secreting cells in a population of splenocytes,determined by the ex vivo ELISPOT assay described above, and generallyas in Bruña-Romero et al. (2001a).

Example 7 Inducing a Long-Lasting Protection Against P. yoelii MalariaInfection by Prime-Boost Regimens with Different AdenovirusSerotype-Based Vaccines

Recombinant Adenovirus serotype 5 expressing a CS antigen of P. yoeliiwas shown to elicit protection when used in prime-boost regimen incombination with a recombinant vaccinia virus carrying the same antigen(Bruña-Romero et al. 2001a). An experiment to investigate the capabilityof prime/boost regimens based on adenovirus vectors carryingcodon-optimized CS and derived from two different serotypes to inducelong-lasting protection against the P. yoelii CS antigen was designed.This study enrolls Balb/C mice distributed in experimental groups of 12mice each. Animals are immunized by intramuscular injection of anoptimal dose of Ad5ΔE3 or Ad35ΔE3 viral vectors carrying either the P.yoelii CS gene (Ad5ΔE3-CS.Pyoel and Ad35ΔE3-CS.Pyoel) or no transgene(Ad5ΔE3-empty and Ad35ΔE3-empty). One group of animals is primed at week0 with Ad5ΔE3-CS.Pyoel and boosted at week 8 with Ad35ΔE3-CS.Pyoel.Another group of mice is primed at week 0 with Ad35ΔE3-CS.Pyoel andboosted at week 8 with Ad5ΔE3-CS.Pyoel. Other groups of mice are primedat week 0 with Ad35ΔE3-CS.Pyoel or Ad5ΔE3-CS.Pyoel and boosted at week 8with the same vector. Finally, a control group of mice is primed at week0 with Ad5ΔE3-empty and boosted at week 8 with Ad35ΔE3-empty. At week 2post-boost, six mice of each group are sacrificed to allow evaluationand characterization of humoral and cellular immune responses withimmunological assays well known to persons skilled in the art. Theremaining six mice from each group are challenged with live sporozoites.The immunization, challenge and read out are scheduled in Table VI.Protection against malaria infection will be monitored and measuredusing assays well known to people skilled in the art as described above.Vaccine regimens based on Ad35 alone or Ad5/Ad35 combinations areexpected to be superior or at least comparable in efficacy as comparedto regimens based solely on Ad5.

Example 8 Inducing a Long-Lasting Immunity Against P. falciparum MalariaInfection by Prime-Boost Regimens with Different AdenovirusSerotype-Based Vaccines

An experiment to investigate the ability of prime/boost regimens basedon adenovirus vectors derived from two different serotypes to inducelong-lasting immunity against the P. falciparum CS antigen was designed.The study enrolls B10.BR mice distributed in experimental groups of 24mice each. Animals are immunized by intramuscular injection of anoptimal dose of adenoviral vectors carrying either the full-length CSgene (Ad5ΔE3-CS.Pfalc and Ad35ΔE3-CS.Pfalc) or the GPI-anchor sequenceminus CS gene (Ad5ΔE3-CS.Pfalc(−28)/(−14) andAd35ΔE3-CS.Pfalc(−28)/(−14)) or no transgene (Ad5ΔE3-empty andAd35ΔE3-empty). One group of animals is primed at week 0 withAd5ΔE3-CS.Pfalc or Ad5ΔE3-CS.Pfalc(−28)/(−14) and boosted at week 8 withAd35ΔE3-CS.Pfalc or Ad35ΔE3-CS.Pfalc(−28)/(−14). Another group of miceis primed at week 0 with Ad35ΔE3-CS.Pfalc or Ad35ΔE3-CS.Pfalc(−28)/(−14)and boosted at week 8 with Ad5ΔE3-CS.Pfalc orAd5ΔE3-CS.Pfalc(−28)/(−14). Another group of mice is primed at week 0with Ad35ΔE3-CS.Pfalc or Ad35ΔE3-CS.Pfalc(−28)/(−14) and boosted at week8 with the same vector. Finally, a control group of mice is primed atweek 0 with Ad5ΔE3-empty and boosted at week 8 with Ad35ΔE3-empty. Atweeks 2 and 6 or 10 or 16 post-boost, six mice are sacrificed at eachtime point and cellular and humoral responses are monitored withimmunological assays well known to persons skilled in the art and asdescribed above. The immunization, challenge and read out are scheduledin Table VII. Vaccine regimens based on Ad35 alone or Ad5/Ad35combinations are expected to be superior or at least comparable inefficacy as compared to regimens based solely on Ad5.

Example 9 Inducing an Immune Response Against the P. falciparum CSAntigen by Prime/Boost Regimens Using Different AdenovirusSerotype-Based Vaccines in Non-Human Primates

An example of an experiment useful to investigate the capability ofprime/boost regimens based on adenovirus vectors derived from twodifferent serotypes to elicit immunity against the P. falciparum CSantigen in non-human primates is described. Moreover, the effect of twodifferent routes of vaccine administration, intramuscular andintradermal, is evaluated.

Rhesus monkeys are vaccinated with adenoviral vectors carrying eitherthe full-length CS gene (Ad5ΔE3-CS.Pfalc or Ad35ΔE3-CS.Pfalc) or theGPI-anchor sequence minus CS gene (Ad5ΔE3-CS.Pfalc(pf-aa-sub) orAd35ΔE3-CS.Pfalc(pf-aa-sub)). Prime/boost regimens (Ad5 followed by Ad35or Ad35 followed by Ad5) are compared to generally applied prime/boostregimens (Ad5 followed by Ad5 or Ad35 followed by Ad35). Humoral andcellular immune responses are monitored using immunological assays wellknown to persons skilled in the art. Serum of immunized monkeys istested by ELISA assay to determine the nature and magnitude of theantibody response against the repeat region of CS. Cellular immuneresponse is measured by ELISPOT assay to determine the amount ofantigen-specific IFN-γ-secreting cells.

TABLE I Names and Genbank database entry numbers of the P. falciparumcircumsporozoite amino acid molecules used to generate the finalconsensus sequence. Wild-type Entry Lab Entry isolates numbers strainsnumbers China AAG37074 3D7 CAA33421 Thailand CAB64171 CAB38998 CSP_PLAFTCSP_PLAFO AAA29542-AAA29552 NP_473175 AAA29555-AAA29576 7G8 CSP_PLAFABrazil CAB64167 C60657 CAB64190-CAB64197 AAA29524 SenegalCAB64180-CAB64189 NF54 AAA29521 Myamar CAB64237-CAB64243 AAA29527 IndiaCAB64169 S05428 Tanzania CAB64168 CSP_PLAFL CAB64170 WELLCOME A54529CAB64172 AAA29554 Gambia AAF03134-AAF03136 CSP_PLAFW A38869 D60657B60657 LE5 CSP_PLAFL B38869 AAA57043 H60657 B29765 Uganda CAA27599CAB64177 Liberia CAB64176 Hondouras CAB64174 South AAA29516-AAA29519East Asia CAB64175 CAB64178 CAB64179

TABLE II Immunization, challenge and read-out schedules for micevaccinations with Ad5.CS.Pyoel (Ad5-PyCS), vp = viral particles permouse. Immunization Viral # ELISPOT/ schedule vector vp mice serumChallenge Prime/challenge Ad5-PyCS 10⁸ 12 2 weeks 2 weeks (6 mice) (6mice) Prime/challenge Ad5-PyCS 10⁹ 12 2 weeks 2 weeks (6 mice) (6 mice)Prime/challenge Ad5-PyCS 10¹⁰ 12 2 weeks 2 weeks (6 mice) (6 mice)Prime/challenge Ad5-empty 10¹⁰ 8 2 weeks 2 weeks (4 mice) (4 mice)

TABLE III Immunization, challenge and read-out schedules for micevaccinations with Ad35.CS.Pyoel (Ad35- PyCS), vp = viral particles permouse. Immunization Viral # ELISPOT/ schedule vector vp mice serumChallenge Prime/challenge Ad35-PyCS 10⁸ 12 2 weeks 2 weeks (6 mice) (6mice) Prime/challenge Ad35-PyCS 10⁹ 12 2 weeks 2 weeks (6 mice) (6 mice)Prime/challenge Ad35-PyCS 10¹⁰ 12 2 weeks 2 weeks (6 mice) (6 mice)Prime/challenge Ad35-empty 10¹⁰ 8 2 weeks 2 weeks (4 mice) (4 mice)

TABLE IV Immunization, challenge and read-out schedules for micevaccinations with Ad5.CS.Pfalc (Ad5-PfCS, with or without anchor), vp =viral particles per mouse. Immunization Viral # ELISPOT/ ELISPOT/schedule vector vp mice serum serum Prime Ad5-PfCS 10⁸ 12 2 weeks 6-8weeks (6 mice) (6 mice) Prime Ad5-PfCS 10⁹ 12 2 weeks 6-8 weeks (6 mice)(6 mice) Prime Ad5-PfCS 10¹⁰ 12 2 weeks 6-8 weeks (6 mice) (6 mice)Prime Ad5-empty 10¹⁰ 8 2 weeks 6-8 weeks (4 mice) (4 mice)

TABLE V Immunization, challenge and read-out schedules for micevaccinations with Ad35.CS.Pfalc (Ad35-PfCS, with or without anchor), vp= viral particles per mouse. Immunization Viral # ELISPOT/ ELISPOT/schedule vector vp mice serum serum Prime Ad35-PfCS 10⁸ 12 2 weeks 6-8weeks (6 mice) (6 mice) Prime Ad35-PfCS 10⁹ 12 2 weeks 6-8 weeks (6mice) (6 mice) Prime Ad35-PfCS 10¹⁰ 12 2 weeks 6-8 weeks (6 mice) (6mice) Prime Ad35-empty 10¹⁰ 8 2 weeks 6-8 weeks (4 mice) (4 mice)

TABLE VI Immunization, challenge and read-out schedules for mice in aprime-boost vaccination set-up using Ad5.CS.Pyoel (Ad5-PyCS) andAd35.CS.Pyoel (Ad35-PyCS). Viral vector- Immuni- Viral boost zationvector- (after # ELISPOT/ schedule prime 8 weeks) mice serum challengePrime-boost/ Ad5-PyCS Ad5-PyCS 12 2 wks post 2 wks post challenge boostboost (6 mice) (6 mice) Prime-boost/ Ad35-PyCS Ad35-PyCS 12 2 wks post 2wks post challenge boost boost (6 mice) (6 mice) Prime-boost/ Ad5-PyCSAd35-PyCS 12 2 wks post 2 wks post challenge boost boost (6 mice) (6mice) Prime-boost/ Ad35-PyCS Ad5-PyCS 12 2 wks post 2 wks post challengeboost boost (6 mice) (6 mice) Prime-boost/ Ad5- empty Ad35- empty 12 2wks post 2 wks post challenge boost boost (6 mice) (6 mice)

TABLE VII Immunization, challenge and read-out schedules for mice in aprime-boost vaccination set-up using Ad5.CS.Pfalc (Ad5-PfCS) andAd35.CS.Pfalc (Ad35-PfCS), with or without GPI anchor. Viral vector-Immuni- Viral boost zation vector- (after # ELISPOT/ ELISPOT/ scheduleprime 8 weeks) mice serum serum Prime-boost/ Ad5-PfCS Ad5-PfCS 12 2 wkspost 6/10/16 challenge boost wks post (6 mice) boost (6 mice)Prime-boost/ Ad35-PfCS Ad35-PfCS 12 2 wks post 2 wks post challengeboost boost (6 mice) (6 mice) Prime-boost/ Ad5-PfCS Ad35-PfCS 12 2 wkspost 2 wks post challenge boost boost (6 mice) (6 mice) Prime-boost/Ad35-PfCS Ad5-PfCS 12 2 wks post 2 wks post challenge boost boost (6mice) (6 mice) Prime-boost/ Ad5-empty Ad35-empty 12 2 wks post 2 wkspost challenge boost boost (6 mice) (6 mice)

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1. A method for generating an immune response against a Plasmodiumfalciparum circumsporozoite (CS) antigen in a subject, the methodcomprising: a) administering to the subject a composition comprising areplication-defective adenovirus comprising a nucleic acid moleculeencoding the protein of SEQ ID NO:6, wherein the nucleic acid moleculeis codon-optimized; and b) further administering to the subject acomposition comprising at least an immunogenic part of a CS protein. 2.The method according to claim 1, wherein the nucleic acid molecule usedin step a) is codon optimized for elevated expression in a mammal. 3.The method according to claim 2, wherein the mammal is a human.
 4. Themethod according to claim 1, wherein the nucleic acid molecule used instep a) comprises SEQ ID NO:4.
 5. The method according to claim 1,wherein the subject is a mammal.
 6. The method according to claim 5,wherein the mammal is a human.
 7. The method according to claim 1,further comprising: c) administering to the subject a second compositioncomprising a recombinant replication-defective adenovirus comprising anucleic acid molecule encoding the protein of SEQ ID NO:6, wherein thenucleic acid molecule is codon-optimized.
 8. The method according toclaim 7, wherein the adenovirus in each composition is of a differentserotype.
 9. The method according to claim 1, wherein thereplication-defective adenovirus is from a serotype selected from thegroup consisting of Ad11, Ad26, Ad34, Ad35, Ad48, Ad49, and Ad50. 10.The method according to claim 9, wherein the replication-defectiveadenovirus is from Ad35.
 11. The method according to claim 1, whereinthe composition comprising at least an immunogenic part of a CS proteinused in step b) comprises purified CS protein.
 12. The method accordingto claim 1, wherein the composition comprising at least an immunogenicpart of a CS protein used in step b) comprises RTS,S.
 13. The methodaccording to claim 1, wherein step b) is performed after step a). 14.The method according to claim 1, wherein step b) is performed beforestep a).
 15. The method according to claim 7, wherein step c) isperformed after step a) and before step b).
 16. The method according toclaim 7, wherein step c) is performed after step a) and wherein step a)is performed after step b).
 17. The method according to claim 7, whereinstep c) is performed after step b) and wherein step b) is performedafter step a).